PMC:1914094 / 14249-16066 JSONTXT

{"project":"WikiPainGoldStandard","denotations":[{"id":"T71","span":{"begin":45,"end":53},"obj":"BINDING"},{"id":"T72","span":{"begin":700,"end":711},"obj":"BINDING"},{"id":"T73","span":{"begin":1298,"end":1309},"obj":"BINDING"},{"id":"T74","span":{"begin":1536,"end":1547},"obj":"BINDING"},{"id":"T75","span":{"begin":1707,"end":1714},"obj":"GENEXPRESSION"},{"id":"T76","span":{"begin":0,"end":4},"obj":"Protein entrezID:943"},{"id":"T77","span":{"begin":5,"end":12},"obj":"Protein entrezID:943"},{"id":"T78","span":{"begin":30,"end":34},"obj":"Protein entrezID:7295"},{"id":"T79","span":{"begin":720,"end":724},"obj":"Protein entrezID:7295"},{"id":"T80","span":{"begin":736,"end":740},"obj":"Protein entrezID:943"},{"id":"T81","span":{"begin":1257,"end":1261},"obj":"Protein entrezID:7295"},{"id":"T82","span":{"begin":1315,"end":1319},"obj":"Protein entrezID:943"},{"id":"T83","span":{"begin":1469,"end":1473},"obj":"Protein entrezID:2745"},{"id":"T84","span":{"begin":1551,"end":1555},"obj":"Protein entrezID:943"},{"id":"T85","span":{"begin":1715,"end":1719},"obj":"Protein entrezID:943"},{"id":"T86","span":{"begin":1520,"end":1523},"obj":"NEGATED"},{"id":"T87","span":{"begin":1703,"end":1706},"obj":"NEGATED"}],"relations":[{"id":"R51","pred":"REPORTED THEME","subj":"T71","obj":"T78"},{"id":"R52","pred":"REPORTED THEME","subj":"T71","obj":"T78"},{"id":"R53","pred":"REPORTED THEME","subj":"T72","obj":"T80"},{"id":"R54","pred":"REPORTED THEME","subj":"T73","obj":"T82"},{"id":"R55","pred":"REPORTED THEME","subj":"T74","obj":"T84"},{"id":"R56","pred":"REPORTED THEME","subj":"T75","obj":"T85"},{"id":"R57","pred":"REPORTED CAUSE","subj":"T76","obj":"T71"},{"id":"R58","pred":"REPORTED CAUSE","subj":"T77","obj":"T71"},{"id":"R59","pred":"REPORTED CAUSE","subj":"T79","obj":"T72"},{"id":"R60","pred":"REPORTED CAUSE","subj":"T81","obj":"T73"},{"id":"R61","pred":"REPORTED CAUSE","subj":"T83","obj":"T74"},{"id":"R62","pred":"REPORTED ","subj":"T86","obj":"T74"},{"id":"R63","pred":"REPORTED ","subj":"T87","obj":"T75"}],"text":"CD30/TNFRSF8 is the principal Trx1 sensitive receptor on various lymphoid cell lines\nTo identify the unknown Trx1 target protein, we performed cell surface trapping on a larger scale (5 × 109 LCL-721.220 cells), purified the Trx1-interacting surface protein by SAv affinity purification and visualized the protein by colloidal Coomassie staining (Figure 3A, left panel). The 160 kDa band was absent in the control precipitation with Trx1(CCAAA). Corresponding bands from non-reducing and reducing lanes were subjected to tryptic digestion and LC-MS/MS analysis. From both samples the unknown protein was identified as TNFRSF8 (CD30), a member of the TNFR superfamily. To validate the direct covalent interaction between Trx1(CSAAA) and CD30, an aliquot of trapped complexes from the same experiment was separated under non-reducing and reducing conditions and subjected to immunoblotting analysis with anti-Trx1 (Figure 3A, middle panel) and anti-CD30 antibodies (Figure 3A, right panel), respectively. The observed mobility difference between non-reducing (NR) and reducing (R) lanes demonstrated the formation of a mixed disulfide conjugate (Figure 3A, right panel). Additional immunoblotting experiments demonstrated that low nanomolar concentrations of Trx1(CSAAA) are sufficient to detect the interaction with CD30 (Figure 3B) and also confirmed that trapping of CD30 depends on the N-terminal cysteine of the CXXC motif (Figure 3C, lanes 1–8). Application of the Grx1 trapping mutant under the same conditions did not lead to its conjugation to CD30 (Figure 3C, lanes 9 and 10). Immunoblotting and flow cytometry experiments confirmed that BL-41 cells, unlike the other lymphocytic cell lines, do not express CD30 (Supplementary Figure S2), thus explaining the absence of the 160 kDa conjugate band (Figure 2B)."}